Cationic bridged stapled peptide and application thereof

A staple and cationic technology, applied in the field of biomedicine, to achieve strong resistance to serum protease hydrolysis, reduce the effect of polypeptide biological activity, and long retention time

Pending Publication Date: 2019-12-31
CHONGQING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these existing methods can only form loops on the hydrophobic side of the polypeptide, and how to construct staple peptides on the hydrophilic side is still a blank

Method used

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  • Cationic bridged stapled peptide and application thereof
  • Cationic bridged stapled peptide and application thereof
  • Cationic bridged stapled peptide and application thereof

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0039] DBU: 1,8-diazabicyclo[5.4.0]undec-7-ene Example 1: Preparation of staple antimicrobial peptide

[0040] The preparation route is as follows, such as figure 1 Shown:

[0041] 1. Preparation of oligopeptide solid-phase resin: Rink-AM resin (loading capacity is 0.35mmol / g, initial loading capacity of each peptide is 200mg), Fmoc-Val-OH is loaded on Rink-AM by standard solid-phase synthesis method AM resin.

[0042] 2. Preparation of lysine protected by o-nitrobenzenesulfonyl group in the side chain: Weigh 4.68g of Fmoc-Lys(Boc)-OH and place it in a 250mL round bottom flask, then measure the solvent CH 2 Cl 2 60mL and 20mL of trifluoroacetic acid (TFA) were added to the round bottom flask, and 10mL of CH 2 Cl 2 Rinse the measuring cylinder that has taken TFA, and transfer the rinsing solution to a round bottom flask, and stir for 1 h. To the obtained intermediate Fmoc-Lys-OH·TFA, add H 2 0: Tetrahydrofuran (THF) = 1:1 (55 mL each), N-ethyldiisopropylamine neutralize...

Embodiment 2

[0059] Embodiment 2: the effect of antimicrobial peptides of staples in inhibiting bacterial growth

[0060] The antibacterial activity detection was carried out according to the conventional doubling dilution method. Add 180uL of bacterial solution and 20uL of drug solution to each well of the first row of 96-well plate, (drug concentration 128ug / mL) set up 3 multiple wells in each well, use TSB medium as negative control, and then doubling dilution to make the final concentration double Decrease, incubate at 37 degrees for 16-20h, measure the absorbance of each well, the lowest concentration with no change in absorbance is the minimum inhibitory concentration (MIC).

[0061] Table 1. Effects of Staple Peptides on Bacterial Growth Inhibition

[0062]

[0063] It can be seen from Table 1 that the staple antimicrobial peptide has broad-spectrum and high-efficiency antibacterial activity, and the staple peptide compound 115 connected with 1,2-dimethylene phenyl is effective ...

Embodiment 3

[0064] Example 3: Staple Antimicrobial Peptide Resistance to Serum Hydrolysis

[0065] Experimental method: the peptides (LH098, LH065, LH115) were incubated with 25% fresh human serum (v / v) at 37°C (the final concentration of the peptide was 1.28 mg / mL). Take 60ul at different time points (0, 1h, 2h, 3h, 6h, 9h, 12h and 24h), then add 120uL of 12% trichloroacetic acid solution (water: acetonitrile 1:3), precipitate serum at 4°C for 30min protein. Then centrifuge at 14000rpm for 10min, take the supernatant, and analyze it by high performance liquid chromatography. See the experimental results figure 2 .

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Abstract

The invention discloses a cationic bridged stapled peptide. The stapled peptide is a cyclic polypeptide with 10-20 amino acid residues, and has the following sequences and structural characteristics:a polypeptide sequence can be shown as a general formula P<n>-K<c>-P<m>-K<c>-P, wherein the P<n>, the P<m> and the P are polypeptide fragments with n, m and i amino acid residues, the n and thei are natural numbers, the m is 3 or 6, the K represents lysine, and the c represents that a cyclic structure is formed between two lysine side chain amino groups. The invention provides a preparationmethod and application of the cationic bridged stapled peptide. A cyclic structure is innovatively formed on one side of the hydrophilic surface of amphiphilic cationic antibacterial peptide, and thestaple antibacterial peptide is prepared. The antibacterial peptide has the following advantages: the antibacterial peptide has broad-spectrum antibacterial activity, and especially has a significantinhibition effect on clinical drug-resistant bacteria; the structure is simple and synthesis is easy; the biological stability is good; the antibacterial peptide can be applied to prevention and treatment of human or animal infectious diseases or tumors.

Description

technical field [0001] The invention designs a class of cation-bridged staple peptides, and also relates to the preparation and application of these polypeptides, belonging to the field of biomedicine. Background technique [0002] Many literatures have shown that cyclic peptides help to enhance the stability of polypeptides and improve the biological activity of polypeptides. There are three main ways to form cyclic peptides: side chain to side chain cross-linking, head-to-tail connection, and side chain to terminal connection. Among them, the side chains of amino acids are connected to each other to form a loop to form a staple peptide. The formation of staple peptide helps to stabilize the secondary conformation of the peptide and improve the binding ability of the peptide to the receptor. The current method for forming staple peptides is mainly through olefin metathesis reaction, cysteine ​​disulfide bonds to form thioethers or amino acid side chains and carboxyl coupl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/08C07K1/06C07K1/04A61K38/10A61P31/04
CPCC07K7/08A61P31/04A61K38/00
Inventor 张金强夏学锋李红胡宇晨
Owner CHONGQING UNIV
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